Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia.

Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased (p<0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria.

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria.

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r(2)=0.68, p<0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.

Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. III. Biocompatibility Of smaller versus standard microcapsules.

Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.

The rat epididymal fat pad as an implantation site for the study of microcapsule biocompatibility: validation of the method.

The study of microcapsule biocompatibility is hindered by their uneven distribution and low recovery when implanted into the peritoneum. We evaluated the use of the rat epididymal fat pad as a microcapsule implantation site for biocompatibility studies. The recovery rate of microcapsules containing 85Sr-labeled microspheres was 99.6 +/- 0.75%. Microcapsules made from the same batch of nonpurified alginate, were injected into both fat pads of male Lewis rats (n = 18) and retrieved 14 days later. A semiquantitative fibrosis score scaled from 0 to 3.0 showed that the pericapsular reaction was uniform throughout a fat pad, and that the results of the two fat pads were equivalent because the null hypothesis of inequivalence was rejected (P < .001). Thus, this method can be used to compare the biocompatibility of microcapsule of differing compositions.

Studies on small (< 300 microns) microcapsules: II--Parameters governing the production of alginate beads by high voltage electrostatic pulses.

The size of microcapsules is a critical parameter in the immunoisolation of islets of Langerhans by microencapsulation. The use of smaller capsules decreases the total implant volume and improves insulin kinetics and oxygen supply. A high voltage electrostatic pulse system was used for the production of small (< 300 microns) alginate beads, the first step of the encapsulation technique. However, islets often protruded from capsules that were too small, further emphasizing the need for a method to control bead size. A study of 7 parameters [electrostatic pulse amplitude (A), duration (D) and wavelength (lambda), pump flow rate (P), needle gauge, alginate viscosity and distance between electrodes] showed that P (r = 0.981, p = 0.003) and lambda (r = 0.988, p = 0.0002) were the principal determinants of bead size. To detect potential interactions between parameters, 270 combinations of different levels of A, D, lambda, and P were studied. A multivariate regression analysis of these data confirmed that P and lambda are the prime determinants of bead size, and showed that a 2-parameter (P, lambda) model could be used to precisely predict bead size (R2 = 0.84), while keeping the application simple. The precision of the predictive model is only slightly improved by the use of additional parameters. The reliability of the data used to elaborate this model was demonstrated (p = 0.6226) by comparing them with a second data set obtained under the same conditions. A third set of experiments confirmed the applicability of the model. This work has major implications on the preclinical application of microencapsulation since it showed that it is possible to predetermine the bead size.